How to Count Adherent Cells with a Hemocytometer

What are adherent cells and how to maintain them

Adherent cells grow in monolayers, attached to the bottom of the flask. When changing growth medium, this is very convenient as you only have to aspirate carefully the old one, and add the same volume of fresh medium (previously warmed up). However, when cells reach confluency (100% of the flask bottom is covered), they need to be detached from the bottom of the flask and resuspended in the correct amount of medium. How do we know which volume to add? The hemocytometer is our friend.

Preparing a cell suspension

To detach the cells, add 1-4mL trypsin (which is widely used as an enzymatic detaching agent) for 1-5 min. To stop the reaction, add a similar volume of medium and mix well. Transfer to centrifuge tubes and centrifuge as stated in the specific cell line protocol. Remove the supernatant and add 10-20mL of fresh medium. Mix well and take a sample.

Loading the haemocytometer

Prepare the haemocytometer with a cover slide on top (making sure that the slide stands firmly on the slide support - add some ethanol if necessary), mix the sample again and introduce meticulously in the space provided in the hemocytometer, between the cover slide and the glass surface. Make sure that the chamber is properly filled, with no air bubbles, and do exactly the same with the other chamber (if necessary).

Counting cells: what to count

Place under the microscope and count the cells in several squares (usually four per chamber, making sure that you keep them symmetric - the most common choice are the four corner squares) remembering to record the counts and the number of squares counted while you count (or when you've finished counting). Make sure that you consistently count the ones that are touching two of the outer lines, and not count the ones that are touching the other two outer lines.

Counting and calculating the cell density

When done, calculate the average number of cells per square and divide that by the volume of a square, which is generally 1/10000 mL (or multiply the average by 10000 mL-1). Multiply by the volume of the cell suspension when you took the sample and divide by the recommended seeding density. You will get the total volume in which you should resuspend, so substracting the 10-20mL you already added gives you the amount you will have to add when you plate.

Resuspending and plating back the cells

Now that you have the cell density and the volume required to reach the recommended plating density, choose an adequately sized plate/T-flask to accommodate the volume calculated in the previous step. Resuspend the 10-20mL of cells you had in the required extra amount of medium and transfer your cell suspension to the plate/T-flask required. Put back in the incubator and remember to check cell density every two to three days, according to the protocol for your particular line. You can also keep track of how fast they grow by measuring the cell density every 12 hours (depending on the duplication time).